Implementing a user‐friendly format to analyze PRRSV next‐generation sequencing results and associating breeding herd production performance with number of PRRSV strains and recombination events

Abstract The open reading frames (ORF)5 represents approximately 4% of the porcine reproductive and respiratory syndrome virus (PRRSV)‐2 genome (whole‐PRRSV) and is often determined by the Sanger technique, which rarely detects >1 PRRSV strain if present in the sample. Next‐generation sequencing (NGS) may provide a more appropriate method of detecting multiple PRRSV strains in one sample. This work assessed the effect of PRRSV genetic variability and recombination events, using NGS, on the time‐to‐low prevalence (TTLP) and total losses in breeding herds (n 20) that detected a PRRSV outbreak and adopted measures to eliminate PRRSV. Serum, lung or live virus inoculation material collected within 3‐weeks of outbreak, and subsequently, processing fluids (PFs) were tested for PRRSV by RT‐qPCR and NGS. Recovered whole‐PRRSV or partial sequences were used to characterize within and between herd PRRSV genetic variability. Whole‐PRRSV was recovered in five out of six (83.3%) lung, 16 out of 22 (72.73%) serum and in five out of 95 (5.26%) PF. Whole‐PRRSV recovered from serum or lung were used as farm referent strains in 16 out of 20 (80%) farms. In four farms, only partial genome sequences were recovered and used as farm referent strains. At least two wild‐type PRRSV strains (wt‐PRRSV) were circulating simultaneously in 18 out of 20 (90%) and at least one vaccine‐like strain co‐circulating in eight out of 20 (40%) farms. PRRSV recombination events were detected in 12 farms (59%), been 10 out of 12 between wt‐PRRSV and two out of 12 between wt‐PRRSV and vaccine‐like strains. Farms having ≥3 strains had a 12‐week increase TTLP versus herds ≤2 strains detected. Farms with ≤2 strains (n 10) had 1837 and farms with no recombination events detected (n 8) had 1827 fewer piglet losses per 1000 sows versus farms with ≥3 PRRSV strains (n 8) or detected recombination (n 10), respectively. NGS outcomes and novel visualization methods provided more thorough insight into PRRSV dynamics, genetic variability, detection of multiple strains co‐circulating in breeding herds and helped establish practical guidelines for using PRRSV NGS outputs.

The PRRSV is an enveloped, single strained positive-sense RNA virus in the order Nidovirales, family Arteriviridae (Kuhn et al., 2016).
Within the whole-PRRSV genome, the specific region ORF5b, also known and herein identified as ORF5, encodes the virus envelope protein and has been extensively used in molecular epidemiology and evolutionary studies to characterize distinct isolates (Brar et al., 2015;Lambert et al., 2019;Larochelle et al., 2003;Paploski et al., 2019;Paploski et al., 2021;Shi et al., 2010;Trevisan et al., 2021b;Wan et al., 2019b). The ORF5 gene comprises 603 nucleotide base pairs (bp) for PRRSV-2 and 606 bp for PRRSV-1, representing approximately 4% of the whole PRRSV genome and has been primarily used in the Americas to classify PRRSV-2 strains according to restriction fragment length polymorphism (RFLP) patterns (Wesley et al., 1998) and genetic lineages (Lambert et al., 2019;Paploski et al., 2019;Paploski et al., 2021;Shi et al., 2010;Wang et al., 2019b), and to charactherize PRRSV strains in diversity studies (Alkhamis et al., 2016;Arruda et al., 2017;Brar et al., 2015;Ramos et al., 2018;Ramírez et al., 2019;Trevisan et al., 2021b). In U.S. veterinary diagnostic laboratories, sequencing of the PRRSV ORF5 region is performed using the Sanger technique (Sanger et al., 1977) which determines the consensus gene sequence in a sample and is not an efficient tool to sequence multiple viruses if more than one PRRSV strain is present . In addition, evaluating only the ORF5 region misses the opportunity to better understand the whole-PRRSV genome characteristics and perform additional investigations, for example, detection of recombination events (Lalonde et al., 2020).
Next-generation sequencing (NGS) is a technique used to sequence the whole-PRRSV genome (Zhang et al., 2017) and has the potential to more thoroughly detect and characterize multiple PRRSV strains if present in the sample. Although NGS seems promising, the use and interpretation of NGS results by veterinary practitioners remains a dilemma (Risser et al., 2021).
The ability of more than one PRRSV-2 strain or a PRRSV-1 and a PRRSV-2 strain to simultaneously infect a piglet has been demonstrated (Dee et al., 1997;Nilubol et al., 2014). Over time, detection of multiple, distinct PRRSV strains in a breed-finish swine herd has been demonstrated by sequencing the PRRSV ORF5 genome region from individual serum and tissue samples (Dee et al., 2001). Anecdotal discussions between practitioners have commonly reported the perception of more than one PRRSV strain simultaneously circulating in a herd. To better support animal health decisions and interventions, there is a need to detect and characterize the presence of multiple PRRSV strains concurrently circulating in a herd and its association with key production performance outcomes.
Recently, processing fluids (PFs), a population sample type composed by the serosanguineous exudate recovered from testicles and tails during processing time, have been described (Lopez et al., 2018;Vilalta et al., 2018), and adopted as part of surveillance programs to detect PRRSV in newborn piglet populations and have also been validated to monitor breeding herds undergoing PRRSV elimination (Trevisan et al., 2019a). PF is currently one of the most used sample type for PRRSV monitoring in the U.S. (Trevisan et al., 2019b). By applying NGS to PFs, it would be expected to recover sequences in genomic regions in addition to ORF5 and detect one or more strains if present in the sample. This study evaluates the feasibility of applying NGS techniques in PF samples to investigate PRRSV genetic variability, detection of multiple, co-circulating virus strains and recombination events and associate NGS results with production outcomes. two samples around ten weeks after the outbreak (week 8-12); (c) post

Overview study design
week 12, all PF samples were tested for PRRSV and stored at −80 • C at the ISU-VDL. After the farm achieved low prevalence, the last two PF samples with a Ct < 30 were retrospectively selected and submitted for PRRSV NGS ( Figure 1).

NGS procedures
Indigenous samples were submitted to NGS. to remove adapters and trim low-quality ends, depleting sequences with lengths of less than 36 nt, and sequencing quality analysis with FastQC (Andrews, 2010). Kraken v0.10.5-β (Wood & Salzberg, 2014) was used to classify cleaned reads. Reads belonged to PRRSV were de novo assembled with SPAdes (v 3.5.0) as described previously (Wang et al., 2019a;Wang et al., 2019b).

Between herd PRRSV genetic comparisons
Recovered near complete-PRRSV sequences representing strains within three weeks from the outbreak from serum, lung or LVI material were used to construct a phylogenetic tree for between participant farms comparison. Sequences were uploaded in Geneious Prime 202.0.1 (https://www.geneious.com), aligned using available MAFFT v7.450 default settings (Katoh, Misawa, Kuma & Miyata, 2002;Katoh & Standley, 2013), and a Neighbor-Joining tree was built using the Tamura-Nei genetic distance model in Geneious Tree Builder using default settings. The phylogenetic tree also included US commercially available PRRS-modified live vaccine sequences for comparison with farm referent strains.
Additionally, structural and non-structural genes (ORF1-7) and non-structural proteins (nsp1-12) were annotated from the ATTC VR 2332 strain (GenBank U87392.3). ORF gene and nsp protein sequences were extracted and pairwise aligned using the MAFFT v7.450 available in Geneious, exported in a FASTA file format, and imported into R (R. Development Core Team, 2020) to extract and plot pairwise distances between different groups, ORF's and nsp's, using the Adegenet package (Jombart, 2008)

Recombination analysis
The near complete-PRRSV genome recovered from serum, lung or LVI material at the time of outbreaks were used to investigate recombination events. Sequences from PRRSV-2 MLV vaccines commercially available in the US and complete or near-complete PRRSV-2 genome sequences available in GenBank (https://www.ncbi.nlm. nih.gov/genbank) and identified from the US were downloaded in a FASTA file format. Downloaded sequences and farm recovered near complete-PRRSV genome were collated into a single database and aligned using MAFFT v7.450 default settings (Katoh et al., 2002;Katoh & Standley, 2013). Recombination events and breakpoints were detected using the RDP4 software (Martin et al., 2015) and were supported by at least six of the seven available default parameters: RDP (Martin & Rybicki, 2000), GENECONV (Padidam et al., 1999), BootScan (Martin et al., 2005), MaxChi (Smith, 1992), Chimaera (Posada & Crandall, 2001), SiScan (Gibbs et al., 2000) and 3SEquation (Boni et al., 2007). The near-complete PRRSV sequences with detected recombinations events were further plotted to confirm and visualize recombinant breakpoints using the SimPlot program (Lole et al., 1999). Due to the usage of indigenous samples for NGS, near complete-PRRSV genome or contigs recovered from PF samples, a population sample type, were not evaluated for recombination events.

Detection of multiple PRRSV strains and within-herd comparisons
Two distinct approaches were used for the detection of multiple PRRSV strains: (a) the default was used when the near complete-PRRSV farm referent strain was recovered; (b) the alternative was used when no whole-PRRSV sequence was recovered at the time of the outbreak.
Using a whole-PRRSV farm referent strain: Contigs or whole-PRRSV sequences recovered from PF samples were compared with the whole-PRRSV farm referent strain using the NCBI nucleotide blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The farm-specific whole-PRRSV genome referent sequence was entered as the query sequence and PF NGS outputs, that is, whole-PRRSV or contigs as the subject sequences for a Blastn search. The hit tables for sequences producing significant alignments were downloaded in a file containing commaseparated values. Vaccine-like contigs were identified by repeating these procedures using the US commercially available PRRS modified live vaccines (MLV) strains, that is, Ingelvac PRRS ® MLV (GenBank AF066183), Ingelvac PRRS ® ATP (GenBank DQ988080), Prime Pac ® PRRS (GenBank DQ779791), Fostera ® PRRS (GenBank AF494042) and Prevacent ® PRRS (GenBank KU131568) as the query sequence.
Contigs were considered farm referent strain or a vaccine-like if the similarity with the query sequence was ≥99%, potentially farm referent strain when had similarity ≥98 and <99%, and classified as wildtype when similarity was <98% with the query sequence. Only contigs having a size equal to the smallest known PRRSV genome region, that is, nsp6 region, which is comprised of 48 bp were used in the analysis. The aligned positions of the contig sequences and their similarity with query sequence were plotted in a colour-coded format using a Microsoft Excel spreadsheet.
No whole-PRRSV farm referent strain available: When no whole-PRRSV genome farm referent strain was available, the contigs recovered from PF at different sampling points were initially compared with the ATCC VR-2332 strain, the PRRSV-2 prototype strain , to determine genome positions. The contigs with the most extensive contiguous nucleotide genome coverage from distinct genome regions, recovered in a sample or from distinct samples, were then selected and re-entered as query sequences on the NCBI tool to be compared with contigs recovered from other sampling events and covering the same genome region as the farm referent contig.
The presence of a second wild-type PRRSV strain was considered when at least one of the two criteria was satisfied: a. The recovered contigs covered the entire length of at least one of the 5'UTR, nsp1-5, nsp7, nsp9, ORF2a, ORF3, ORF4, ORF5, ORF6, ORF7 or 3'UTR genome regions and had <98% similarity with the farm referent strain. Genome regions were determined by annotating contigs from the ATCC VR-2332 strain in Geneious. b. Contigs recovered from different genome regions and having a length equal to or greater than the smallest described functional PRRVS genome structure, that is, nsp6 comprised of 48 bp with <98% similarity with the farm referent strain recovered at different sampling points covering similar or distinct genome regions.

Association with production performance
The total number of strains detected in a herd and identification of recombination events were respectively evaluated for the association with TTLP, TTBP and TLs.
TTLP across groups was compared using Kaplan-Meier survival analysis (Kaplan-Meier) available on PROC Lifetest procedure in SAS 9.4 (SAS Institute Inc., Cary, NC). It was measured as the time in weeks it took for the farms to achieve low prevalence after the outbreak.
Farms that dropped the study due to depopulation before 10 weeks post PRRSV outbreak were excluded from the analysis and, after 10 weeks, were censored. Between group, survival differences were determined by using a log-rank test at a .05 significance level.
TTBP was measured as the time in weeks it took for the farms to recover the levels of pigs weaned per week as prior to the outbreak (Linhares et al., 2014). Exponential weighted moving average (EWMA) available in the PROC MACONTROL procedure from SAS 9.4 (SAS Institute, Inc., Cary, NC) was used to scan the database for changes from a baseline level. EWMA parameters were set to 3 sigmas and lambda of 0.4 (Linhares et al., 2014). The number of sigmas adjusts the width of upper and lower control limits, and lambda adjusts the assigned weight to the most recent variable value included in the data (Montgomery, 2009). Baselines were constructed based on the 26weeks previous to the initial PRRS outbreak to cover one complete breeding and two weaning cycles. The number of pigs weaned were considered outside of monitored parameters or returned to baseline if they were outside or returned to monitoring baseline parameters for at least two consecutive weeks (Linhares et al., 2014). TTBP across different groups was compared using the Kaplan-Meier survival estimates by using a log-rank test at a .05 significance level.
TLs were calculated as the total number of pigs not weaned per 1000 sows from the PRRSV outbreak to when the herd returned to TTBP. TL was calculated as the cumulative sum of the number of pigs not weaned below baseline levels prior to the outbreak. TLs across different groups were compared using a generalized linear mixed model available in PROC GLIMMIX procedure in SAS using recombination and number of strains as explanatory variables and TLs as the dependent variable at a .2 significance level.
During the investigation period, no farm exclusion occurred since farms enrolled in this study did not experience outbreaks with other infections pathogen, for example, porcine epidemic diarrhoea virus.

RESULTS
This study enrolled breeding herds (n = 20) that experienced a PRRSV outbreak and adopted measures to eliminate the virus from the herd.
Herds were located in five US states and were linked to eight production systems or veterinary clinics. Breeding herds had a median of 3065 productive sows (25th percentile 2475 and 75th percentile 3290). RT-qPCR or NGS detected only PRRSV-2.
A near complete-PRRSV genome was recovered in five out of six  Table S1 for each gene and non-structural proteins.
In addition to the whole-genome sequences of 16 farm referent strains, a large contig (12,888 bp) recovered from PF sample of farm 1 was also included for investigation of recombination events.
Recombination events between different PRRSV wild-type strains were detected in 10 out of 17 (58.8%) farms. Recombination events involving two wild-type strains and a PRRS MLV vaccine strain were detected in two out of 17 (11.8%) farms. Seven of 12 (58.3%) detected F I G U R E 4 Pairwise comparison for farm PRRSV referent strains. Overall, whole-PRRSV genome nucleotide identity is represented by the bar designated WGS. Non-structural proteins nsp1-nsp8 are subregions of the ORF1a gene. The nsp8 is a subregion within the nsp9 codified in the ORF1a gene. Non-structural proteins nsp9 (except nsp8 subregion), nsp10-nsp12 are ORF1b gene subregions. The ORF2b gene is an ORF2a gene subregion. ORF5b gene is also known as ORF5. The ORF5a is an ORF5 gene subregion F I G U R E 5 Similarity plot analysis using a farm referent strain as query sequence against two wild-type Lineage 1A (L1A) PRRSV strains. PRRSV genome regions are presented at the top of the plot. Green and blue boxes outline the recombination breakpoints and genome regions that were derived from each of the parental and minor strains. Genome regions were plotted in Geneious and recombination events determined using SimPlot recombination events were encountered in farms having ≥3 PRRSV strains (Table 1).
Recombination breakpoints were identified at different genomic regions. As examples, Figure 5 showed a recombination format between two Lineage 1A RFLP 1-7-4 wild-type strains, and Figure 6 presented a recombination involving two Lineage RFLP 1-7-4 wildtype strains and a PRRS MLV vaccine strain. In Figure 5, the recombination breakpoint was at the end of the nsp12 protein. In Figure 6, distinct wild-type recombination breakpoints were identified in the ORF1a and    before the farm achieved low prevalence at week 54, recovered contigs from a second wild-type and Fostera-like strain (Figure 9).

Case D:
A 3900 sow farm operating on a continuous breedingfarrowing system, AASV classification Positive Unstable (I) (Holtkamp et al., 2011), faced a PRRS outbreak with a Lineage 1C RFLP 1-2-4 PRRSV strain. The farm adopted exposure to sows and gilts with Ingelvac ATP, an attenuated PRRSV vaccine, during the week of the outbreak. Replacement animals were brought from an external source and exposed to Ingelvac ATP twice.

F I G U R E 7
Comparison between a farm-specific wild-type PRRSV strain and genome fragments, that is, contigs, recovered from processing fluid samples characterizing the presence of at least five PRRSV strains. The structure at the top of the chart represents the farm referent strain position of open reading frames (ORFs) and non-structural proteins (nsp). The percentage of nucleotide identity between contig and farm referent strain is labelled at the center of each contig. Colours represent the level of similarity with farm referent strain or with a commercially available PRRSV MLV vaccine. Contigs in green: >99%; in orange: ≥98 and <99%; in light red: <99% similarity with farm referent strain; in blue: >99% similarity Fostera; in yellow: >99% similarity with Ingelvac MLV; and in dark red: new PRRSV introduction F I G U R E 8 Comparison between a farm-specific wild-type PRRSV strain and genome fragments, that is, contigs, recovered from processing fluid sample characterizing the presence of at least 4 PRRSV strains. The structure at the top of the chart represents the farm referent strain position of open reading frames (ORFs) and proteins (nsp). The percentage of nucleotide identity between each contig and farm referent strain is labelled at the center of each contig. Colours represent the level of similarity with farm referent strain or with a commercially available PRRSV MLV vaccine. Contigs in green: >99%; in orange: ≥98 and <99%; in red: <99% similarity with farm referent strain; in blue: >99% similarity with Fostera; in purple: >99% similarity with Prevacent

F I G U R E 9
Comparison between a farm-specific wild-type referent PRRSV strain and genome fragments, that is, contigs, recovered from processing fluids samples characterizing the presence of at least three PRRSV strains.  Table 2.

DISCUSSION
This study used 20 breeding herds that faced a PRRSV outbreak and adopted measures to eliminate the virus from the herd; those were followed over time and had PF samples submitted for PRRSV NGS.
Results provided novel insights into farm-level PRRSV detection, the occurrence of multiple strains circulating in a production system, and  of multiple similar PRRSV strains may have prevented the recovery of near complete-PRRSV genomes in PF. When two genetically similar PRRSV-2 (Zhang et al., 2017) or PRRSV-1 (Vandenbussche et al., 2021) were present in the same sample submitted to NGS for wholegenome sequencing, the recovery of contigs (i.e., genome fragments) of each virus strain was successful, and the consensus whole-genome sequence was obtained; however, the recovery of a near complete-PRRSV genome (∼15.1 kb) for each virus strain present in the sample has failed due to the short read length sequencing method and the inability to correctly align contigs with their respective viruses.
Although this limitation occurred, it can be frequently based on the concentration of virus in the sample or sample type; the recovery of PRRSV genome fragments has provided valuable, scientific evidence to support the presence of multiple PRRSV strains simultaneously circulating in breeding herds.
The novel proposed approach of comparing contigs described in this report has provided valuable insights into PRRSV genetic diversity in breeding herds and the dynamic epidemiology that complicates the ability to control the virus effectively. Prior to using NGS, the presence of multiple PRRSV strains in herds could have gone undetected or remained unknown using traditional diagnostic methods and ORF5, that is, Sanger sequencing, which only provides the consensus sequence of the target gene.
Recently, the concept of applying NGS directly to field samples has been explored (Lalonde et al., 2020). There have been encouraging reports describing the potential recovery of a near complete-PRRSV genome from field clinical serum samples with Ct up to 34.1, oral fluid with Ct up to 32.8 and tissue and lung samples with Ct up to 26.5 (Gagnon et al., 2021). RNA viruses, such as PRRSV, are challenging to sequence and characterize using high-throughput sequencing technologies due to their genetic diversity, relatively short genome lengths and lack of conserved genome regions (Fitzpatrick et al., 2021). In this study, we have demonstrated the use of the Illumina MiSeq platform for high-throughput sequencing output, and our results agreed with previous reports that near complete-PRRSV genome sequences should not be expected for lung and serum samples with Ct > 25 (Zhang et al., 2017). Nevertheless, it is possible that using other commercially available high-throughput sequencing platforms, long read-length sequencing methods, and library sequence preparation kits may result in different levels of success of whole-PRRSV genome recovery (Reuter et al., 2015).
Following the proposed criteria described in this report, we demonstrated the presence of multiple PRRSV strains simultaneously circulating in a breeding herd at different sampling points. These findings were consistent in different herds. NGS results are designed to provide the whole genome, not only the ORF5 region, and provide better insights into PRRSV genetic diversity, the number of strains circulating in breeding herds and its molecular characteristics. Sanger gene sequencing techniques (Sanger et al., 1977) are likely to be more successful in sequencing an entire gene, for example, ORF5 or ORF7; however, these traditional sequencing methods lack the ability to detect more than one sequence in a sample consistently but may represent a consensus sequence of the gene of interest (Harmon et al., 2019).
It has been previously reported that when more than one PRRSV strain is circulating in a population, the more aggressive strain with a higher replication rate will suppress the replication of the alternate virus, which ultimately represents the less predominant virus in a herd (Brar et al., 2015). The high replication rate varies due to a wide range of cell tropism   (Christianson et al., 1993;Nielsen et al., 2002;Terpstra et al., 1991). Adopting biomanagement and biocontainment procedures , such as McREBEL (McCaw, 2000), should be encouraged to help minimize PRRSV transmission to other piglets and rooms, reducing potential recirculation of multiple strains on the farm, and perhaps lessen the PRRSV impact in the downstream flow. It is also possible that PRRSV outbreaks occurring in the downstream flow, attributed as lateral introductions, that is, from another pig farm, could potentially be leaking the less predominant PRRSV strains circulating in the breeding herd.
PRRSV recombination events, or mutations that occur due to the low fidelity of RNA virus replication, may lead to the emergence of new PRRSV strains (Yuan et al., 1999;Zimmerman et al., 2019). Here, we observed that herds with evidence of ≥3 PRRSV strains had most of the detected recombination events described in this study and may indicate a greater likelihood of recombination occurring in farms with ≥3 or more strains co-circulating. Nevertheless, this work adopted the terminology vaccine-like to describe contigs having similarity ≥99% to the commercially available MLV vaccines. As MLV vaccines are preceded by attenuating wild-type strains, it is possible that some contigs could be part of a wild-type strain. No contigs comparison with publicly available databased was made to rule this out.
More than half of enrolled herds had a recombination event detected that involved multiple wild-type strains, including different genome regions. Due to the restricted number of complete or near complete-PRRSV genomes available in GenBank, there is a potential to miss detection of recombination events between wild-type strains.
Alternatively, PRRSV MLV vaccine strains are well characterized, and the identification of recombination events between wild-type and vaccine strains are more likely to occur (Risser et al., 2021;Wang et al., 2019a). In this study, we identified two farms that have been affected by a recombinant PRRSV that involved a wild-type and vaccine strain.
No outbreak investigation was conducted to discern the potential connection between those farms. NGS complete or near-complete PRRSV genome outputs from individual indigenous lung and serum samples were used to investigate recombination events. Even though this study included one large contig obtained from processing a fluid sample in the recombination analysis, a prior plaque purification of PF sample could potentially help to isolate the virus and obtain a complete or near-complete PRRSV genome. There is a potential for recombination events detected from a genome recovered from an indigenous population sample, for example, PF, oral fluid, to be an NGS artifact formed by distinct portions of two or more PRRSV strains if present in the sample.
NGS outputs have a competitive advantage when compared with ORF5 sequencing regarding the ability to better characterize PRRSV strains. For example, the strains presented in Figures 5 and 6 have evidence of being derived from similar ancestors, but in Figure 6, there was also recombination involving an MLV vaccine. Limited genetic comparison to one genome region may prevent the identification of relevant genetic characteristics of the strain. This study enrolled 20 breeding herds and revealed that multiple PRRSV circulating in a production system, that is, ≥3 PRRSV strains, was negatively associated with TTLP and TL and that recombinant strains were also negatively correlated with TTLP. It is possible that individuals cannot develop immunity for all the strains simultaneously, and subpopulations can maintain distinct PRRSV strains circulating on the farm and affecting animal performance.
Even though the use of NGS results to monitor PRRSV detection and genetic diversity is still evolving, a general recommendation involving this diagnostic technique would be to target the recovery of a farmspecific whole-PRRSV or near complete-PRRSV genome at the time of the outbreak. Recovered strains can be used as farm referent strains for further comparison to help identify if new or genetically similar strains are circulating or were introduced into the herd, which helps populate a farm or production system-specific library. For the initial NGS sequencing, samples that are more likely to yield higher viral concentrations, that is, low Ct values, for example, serum and lung, are preferred to help improve the success of acquiring the whole-genome PRRSV sequence. Subsequent NGS can be applied to population samples like PF where further comparisons of recovered sequences, even if genome fragments, with the farm referent strain, is valuable and can help identify and characterize additional PRRSV strains circulating in a breeding herd.